Use of Burkholderia Formulations, Compositions and Compounds to Modulate Crop Yield and/or Corn Rootworm Infestation

ABSTRACT

Provided is the use of or compositions or formulations comprising Burkholderia species, filtrate, supernatant, extract, pesticidally active compound or metabolite derived therefrom as an insecticide, particularly against infestation of Corn Rootworm larvae, and/or as crop yield enhancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 16/177,641 filed on Nov. 1, 2018, which is aContinuation Application of U.S. patent application Ser. No. 15/339,304filed on Oct. 31, 2016, now U.S. Pat. No. 10,149,480, which is aDivisional Application of U.S. patent application Ser. No. 14/548,824filed on Nov. 20, 2014, now U.S. Pat. No. 9,526,251, which is aContinuation-in-Part application of U.S. patent application Ser. No.14/336,601 filed on Jul. 21, 2014, now U.S. Pat. No. 9,433,218, which isa Continuation Application of U.S. patent application Ser. No.13/843,971 filed on Mar. 15, 2013, now U.S. Pat. No. 8,822,193, which isa Continuation-in-Part application of U.S. patent application Ser. No.13/034,575 filed on Feb. 24, 2011, now U.S. Pat. No. 9,701,673, whichclaims the benefit of priority under 37 U.S.C. 119(e) of provisionalapplications 61/308,287 and 61/406,541, filed on Feb. 25, 2010 and Oct.25, 2010, respectively. All of the applications are hereby incorporatedby reference in their entirety.

TECHNICAL FIELD

Provided is the use of or compositions or formulations comprisingBurkholderia species, filtrate, supernatant, extract, pesticidallyactive compound or metabolite derived therefrom as an insecticide,particularly against infestation of Diabrotica spp. (Corn Rootworm)and/or as a crop yield enhancer.

BACKGROUND

Commercial crops are often the targets of attack by insects. Insect canhave a significant negative effect on crop yield. Chemical insecticideshave been effective in eradicating insect infestations; however, thereare disadvantages to using chemical insecticides. Chemical insecidalagents are not selective and may exert a negative effect on beneficialinsects and other organisms as well as the targeted insect. Chemicalinsectidal agents may persist in the environment and generally are slowto be metabolized, if at all. They accumulate in the food chain, andparticularly in the higher predator species, where they can assertnegative effects. Accumulations of chemical insectidal agents alsoresults in the development of resistance to these chemical tools.

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is amajor pest of corn in the United States. The western corn rootwormoverwinters in the egg stage in fields where corn was grown the previousseason. The eggs hatch from late May through June. Thereafter, thelarvae pass through 3 larval stages, or instars, feeding upon the cornroot system. Following completion of larval development, the larvaetransform into pupae, which are white and immobile. Adults of westerncorn rootworms begin to emerge in early July and continue to emerge fromthe pupae stage into August. Adult beetles feed on the corn foliage andsilk. Female beetles lay the vast majority of their eggs in the soil ofcornfields during August and early September. Western corn rootwormlarvae can survive only on corn and a few other species of Poaceae(Branson and Ortman, J. Econ. Entomol. 60: 201-203 (1967); Branson andOrtman, J. Econ. Entomol. 60: 201-203 (1967)). Larval root feedingdecreases plant vigor by reducing the water and nutrients supplied tothe developing corn plants. Extensive root damage weakens the rootsystem and makes the plants more susceptible to lodging (plants leanover or elbow), which eventually reduces corn yield and often results indeath of the plant. Lodged plants are difficult to harvest resulting infurther yield losses. The western corn rootworm adults feed upon cornleaves, which can slow plant growth and, on rare occasions, kill plantsof some corn varieties. The western corn rootworm cause economic lossesthroughout the Midwest and in certain eastern and northeastern stateswhere corn is produced.

Control of corn rootworms has been partially addressed by crop rotation.However, economic demands on the utilization of farmland restrict theuse of crop rotation. In addition, the spread of at least one strain ofrootworm has been documented in which female oviposition occurs insoybean fields, which further complicates crop rotation strategies.Therefore, chemical insecticides are relied upon most heavily toguarantee the desired level of control. Over $250 million worth ofinsecticides are applied annually to control corn rootworms alone in theUnited States. Even with insecticide use, rootworms still can cause over$750 million worth of crop damage each year. The use of chemicalinsecticides to control corn rootworm has several drawbacks. Continualuse of insecticides has allowed resistant insects to evolve. Situationssuch as extremely high populations of larvae, heavy rains, and impropercalibration of insecticide application equipment can result in poorcontrol. Chemical insecticides used for corn rootworm control oftenraises environmental concerns such as contamination of soil and of bothsurface and underground water supplies, because many of them are toxicto humans, wildlife and other nontarget species. As a result, muchresearch has been concentrated in the area of biopesticides.

Thus, there is a need for alternative methods for controlling oreradicating insect infestation on or in plants; methods which areselective, environmentally safe, non-persistent, biodegradable, and thatfit well into pest resistance management schemes.

Disclosed herein is a non-Burkholderia cepacia complex, non-Burkholderiaplantari, non-Burkholderia gladioli, Burkholderia sp., in particular,Burkholderia A396 sp. nov. rinojensis (NRRL Accession No. B-50319,a.k.a., 206) that can decrease Diabrotica infestation and/or increasecrop yield. It is non-pathogenic to vertebrates, such as birds, mammalsand fish, as is described in, for example, U.S. Pub. No. 2011-0207604and U.S. application Ser. No. 13/843,971. Methods of growing theBurkholderia sp. and making a composition of Burkholderia sp. and itsproducts (e.g., a whole-cell broth) are also described in U.S. Pub. No.2011-0207604 and U.S. application Ser. No. 13/843,971.

SUMMARY

Provided herein is a method for increasing yield in Zea mays and/ordecreasing infestation of corn rootworm larvae in a location wheremodulation is desired comprising applying an amount of (a) a culture,suspension or whole cell broth comprising a strain of Burkholderia sp.,or supernatant, filtrate, cell fraction, extract and/or one or morecompounds derived from said culture, suspension or whole cell broth and(b) optionally at least one of a carrier, diluent or adjuvant effectiveto modulate said infestation of corn rootworm larvae at said location.In a particular embodiment, the strain has the identifyingcharacteristics of Burkholderia A396 (NRRL Accession No. B-50319).

Infestation of Diabrotica (corn rootworm) larvae can be modulated bymodulating mortality of corn rootworm larvae, specifically by modulatingand particularly increasing or boosting mortality of corn rootwormlarvae and/or by decreasing hatching rate of eggs laid and/or decreasingthe number of eggs laid in a particular location.

Zea mays yield, measured in Bu/A, can also be modulated by theapplication of Burkholderia sp. culture.

In one aspect, the present disclosure describes a first embodimentrelating to a method for modulating infestation of Diabrotica spp. (cornrootworm) in a location where modulation is desired comprising applyingan effective amount of (a) a culture, suspension or whole cell brothcomprising a strain of Burkholderia sp., or supernatant, filtrate, cellfraction, and/or extract derived from said culture, suspension or wholecell broth, and (b) optionally at least one of a carrier, diluent oradjuvant, to modulate said infestation of corn rootworm at saidlocation.

The method according to embodiment 1, wherein the location wheremodulation is desired is on a plant, plant seed, plant roots, plantpart, seedling or substrate for growing said plant.

The method according to embodiment 1, wherein said Burkholderia sp. isBurkholderia A396 strain.

The method according to embodiment 1, wherein said Burkholderia sp. hasthe identifying characteristics of (NRRL Accession No. B-50319).

The method according to embodiment 1 wherein said Diabrotica (cornrootworm) is Western or Northern Corn Rootworm.

The method according to embodiment 1, wherein said Diabrotica (cornrootworm) is modulated by modulating the mortality of said cornrootworm.

The method according to embodiment 1, wherein the mortality ofDiabrotica (corn rootworm) is modulated and wherein there is a mortalityof corn rootworm of at least about 50% at said location.

The method according to embodiment 1, wherein the wherein the mortalityof corn rootworm is modulated and wherein there is a mortality ofDiabrotica (corn rootworm) of at least about 50% following applicationof said culture, suspension or whole cell broth comprising a strain ofBurkholderia sp., or supernatant, filtrate, cell fraction, extractand/or one or more compounds derived from said culture, suspension orwhole cell broth and (b) at least one of a carrier, diluent or adjuvantat said location.

The method according to embodiment 1, which further comprises applyinganother natural or artificial insecticidal substance.

The method according to embodiment 1, wherein the infestation ofDiabrotica (corn rootworm) is modulated by modulating the rate ofhatching of eggs.

The method according to embodiment 1, wherein said culture, suspensionor whole cell broth comprising a strain of Burkholderia sp., orsupernatant, filtrate, cell fraction, and/or extract derived from saidculture, suspension or whole cell broth further comprise at least one ofa carrier, diluent or adjuvant is formulated into a composition.

Yet in another aspect, the present disclosure relates to a method formodulating plant yield and Diabrotica infestation contemporaneouslycomprising applying an effective amount of a culture, suspension orwhole cell broth comprising a strain of Burkholderia sp., orsupernatant, filtrate, cell fraction, and/or extract derived from saidculture, suspension or whole cell broth to a plant or plant part.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts trial two, with increased Zea mays yield, measured inBu/A at 15% moisture, by applying Burkholderia sp. culture (MBI-206) onDKC52-43VT3 corn hybrid. Y-axis is Bu/A. SDTR is seed treatment withTechnical grade active ingredients. IF is in-furrow. RW is row feet.Means sharing the same letters are not statistically significant(P<0.05)

FIG. 2 depicts trial three, with increased Zea mays yield, measured inBu/A at 15% moisture, by applying Burkholderia sp. culture (MBI-206) onPioneer 1023AM corn hybrid. Y-axis is Bu/A. SDTR is seed treatment withTechnical grade active ingredients. IF is in-furrow. RW is row feet.Means sharing the same letters are not statistically significant (P<0.1)

FIG. 3 depicts trial four, with increased Zea mays yield, measured inBu/A at 15% moisture, by applying Burkholderia sp. culture (MBI-206) onDKC Smart Stax corn hybrid. Y-axis is Bu/A. SDTR is seed treatment withTechnical grade active ingredients. IF is in-furrow. RW is row feet.Means sharing the same letters are not statistically significant(P<0.05)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

While the compositions and methods heretofore are susceptible to variousmodifications and alternative forms, exemplary embodiments will hereinbe described in detail. It should be understood, however, that there isno intent to limit the invention to the particular forms disclosed, buton the contrary, the intention is to cover all modifications,equivalents, and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit a value, unless the contextclearly dictates otherwise, between the upper and lower limit of thatrange and any other stated or intervening value in that stated range, isincluded therein. Smaller ranges are also included. The upper and lowerlimits of these smaller ranges are also included therein, subject to anyspecifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

As defined herein, “derived from” means directly isolated or obtainedfrom a particular source or alternatively having identifyingcharacteristics of a substance or organism isolated or obtained from aparticular source. In the event that the “source” is an organism,“derived from” means that it may be isolated or obtained from theorganism itself or culture broth, suspension or medium used to cultureor grow said organism. A compound “derived from” or “obtainable from”means that a compound may be isolated from or produced by a cellculture, whole cell broth, suspension, filtrate, supernatant, fractionor extract.

As defined herein, “whole broth culture” or “whole cell broth” refers toa liquid culture containing both cells and media. If bacteria are grownon a plate, the cells can be harvested in water or other liquid, wholeculture. The terms “whole broth culture” and “whole cell broth” are usedinterchangeably.

As defined herein, “supernatant” refers to the liquid remaining whencells grown in broth or are harvested in another liquid from an agarplate and are removed by centrifugation, filtration, sedimentation, orother means well known in the art.

As defined herein, “filtrate” refers to liquid from a whole brothculture that has passed through a membrane.

As defined herein, “extract” refers to liquid substance removed fromcells by a solvent (water, detergent, buffer, organic solvent) andseparated from the cells by centrifugation, filtration or other method.

As defined herein, “metabolite” refers to a compound, substance orbyproduct of a fermentation of a microorganism, or supernatant,filtrate, or extract obtained from a microorganism that has pesticidalactivity.

As defined herein, an “isolated compound” is essentially free of othercompounds or substances, e.g., at least about 20% pure, preferably atleast about 40% pure, more preferably about 60% pure, even morepreferably about 80% pure, most preferably about 90% pure, and even mostpreferably about 95% pure, as determined by analytical methods,including but not limited to chromatographic methods, electrophoreticmethods. A compound “derived from” a Burkholderia sp. species alsoencompasses a metabolite.

As defined herein, “carrier” is an inert, organic or inorganic material,with which the active ingredient is mixed or formulated to facilitateits application to plant or other object to be treated, or its storage,transport and/or handling.

The term “diluent” is intended to mean an aqueous or non-aqueoussolution with the purpose of diluting the active ingredient.

As defined herein, “modulate”, is used to mean to alter the amount of avalue. For example, decrease in rate of insect infestation or increasein Zea Mays yield.

As defined herein, “pest infestation”, is the presence of a pest in anamount that causes a harmful effect including an insect such as cornrootworm, a disease or infection in a host population, or emergence ofan undesired weed in a growth system.

As defined herein “pesticide”, is a substance derived from a biologicalproduct or chemical substance that increase mortality or inhibit thegrowth rate of plant pests and includes but is not limited tonematicides, algaecides, herbicides, insecticides, plant fungicides,plant bactericides, and plant viricides.

The method disclosed herein can comprise or be derived from an organismhaving the identifying characteristics of a Burkholderia species, moreparticularly, from an organism having the identifying characteristics ofa strain of Burkholderia A396 (NRRL Accession No. B-50319), oralternatively from any other microorganism. The methods comprisecultivating these organisms and obtaining the compounds and/orcompositions by isolating these compounds from the culture of theseorganisms.

In particular, the organisms are cultivated in nutrient medium usingmethods known in the art. The organisms can be cultivated by shake flaskcultivation, small scale or large scale fermentation (including but notlimited to continuous, batch, fed-batch, or solid state fermentations)in laboratory or industrial fermenters performed in suitable medium andunder conditions allowing cell growth. The cultivation can take place insuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable can be available from commercial sources or prepared accordingto published compositions.

After cultivation, the compounds, metabolites and/or compositions can beextracted from the culture broth. The extract can be fractionated bychromatography. Compounds used can be metabolites and in a specificembodiment may include but is not limited to compounds set forth in USPatent Application Publication No. US20110207604, US20140073501 and PCTappln. no. PCT/US2014/015799, the contents of all which are publishedand incorporated herein by reference in their entirety.

The substances set forth above used in the compositions and methodsdisclosed herein can be formulated in any manner. Non-limitingformulation examples include but are not limited to Emulsifiableconcentrates (EC), Wettable powders (WP), soluble liquids (SL),Aerosols, Ultra-low volume concentrate solutions (ULV), Soluble powders(SP), Microencapsulation, Water dispersed Granules, Flowables (FL),Microemulsions (ME), Nano-emulsions (NE), and Seed treatments etc. Inany formulation described herein, percent of the active ingredient iswithin a range of 0.01% to 99.99%.

The compositions can be in the form of a liquid, gel or solid. Liquidcompositions comprise pesticidal compounds derived from a Burkholderiasp strain, e.g. a strain having the identifying characteristics ofBurkholderia A396 (NRRL Accession No. B-50319).

A solid composition can be prepared by suspending a solid carrier in asolution of pesticidal compounds and drying the suspension under mildconditions, such as evaporation at room temperature or vacuumevaporation at 65° C. or lower.

A composition can comprise gel-encapsulated compounds derived from theBurkholderia strain. Such gel-encapsulated materials can be prepared bymixing a gel-forming agent (e.g., gelatin, cellulose, or lignin) with aculture or suspension of live or inactivated Burkholderia sp, or acell-free filtrate or cell fraction of a Burkholderia culture orsuspension, or a spray- or freeze-dried culture, cell, or cell fractionor in a solution of pesticidal compounds used in the method of theinvention; and inducing gel formation of the agent.

The composition can additionally comprise a surfactant to be used forthe purpose of emulsification, dispersion, wetting, spreading,integration, disintegration control, stabilization of activeingredients, and improvement of fluidity or rust inhibition. In aparticular embodiment, the surfactant is a non-phytotoxic non-ionicsurfactant which preferably belongs to EPA Inerts List 4B. In anotherparticular embodiment, the nonionic surfactant is polyoxyethylene (20)monolaurate. The concentration of surfactants may range between 0.1-35%of the total formulation, preferred range is 5-25%. The choice ofdispersing and emulsifying agents, such as non-ionic, anionic,amphoteric and cationic dispersing and emulsifying agents, and theamount employed is determined by the nature of the composition and theability of the agent to facilitate the dispersion of the compositions ofthe present invention.

The composition as set forth above also comprises a stabilizing agent,which stabilizes a biological pesticide composition against physicalseparation and loss of activity due to exposure to sunlight. Thisstabilizing agent can be a benzoic acid salt or lignin sulfonate salt.

The composition as noted can further comprise an insecticide. Theinsecticide may include but is not limited to avermectin, Bt, neem oil,spinosads, Burkholderdia sp. as set forth in US Patent Appln. Pub. No.2011-0207604, entomopathogenic fungi such a Beauveria bassiana andchemical insecticides including but not limited to organochlorinecompounds, organophosphorous compounds, carbamates, pyrethroids, andneonicotinoids.

The composition of the present disclosure contains Burkholderia sp. andcan be combined with another microorganism and/or pesticide (e.g.,nematicide, fungicide, insecticide, herbicide). The microorganism caninclude, but is not limited to, an agent derived from Bacillus sp.(e.g., B. firmus, B. thuringiensis, B. pumilus, B. licheniformis, B.amyloliquefaciens, B. subtilis), Paecilomyces sp. (P. lilacinus),Pasteuria sp. (P. penetrans), Pseudomonas sp., Brevabacillus sp.,Lecanicillium sp., Ampelomyces sp., Pseudozyma sp., Streptomyces sp. (S.bikiniensis, S. costaricanus, S. avermitilis), Trichoderma sp.,Gliocladium sp., avermectin, Myrothecium sp., Paecilomyces spp.,Sphingobacterium sp., Arthrobotrys sp., Chlorosplrnium sp, Neobulgariasp, Daldinia sp, Aspergillus sp, Chaetomium sp, Lysobacter sp, Lachnumpapyraceum, Verticillium suchlasporium, Arthrobotrys oligospora,Pochonia chlamydosporia (synonym: Verticillium chlamydosporium),Hirsutella minnesotensis, Hirsutella rhossiliensis, Pleurotus ostreatus,Omphalotus olearius, Lampteromyces japonicas, Brevudimonas sp., andMuscodor sp.

The pesticide can be a natural oil, oil product or chemical pesticide.In particular, the agent can be a natural oil or oil-product havingnematicidal, fungicidal and/or insecticidal activity (e.g., paraffinicoil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil(including but not limited to bitter orange, orange, lemon) rosemaryoil, pyrethrum, allspice, bergamot, blue gum, camomile, citronella,common jasmine, common juniper, common lavender, common myrrh, fieldmint, freesia, gray santolina, herb hyssop, holy basil, incense tree,jasmine, lavender, marigold, mint, peppermint, pot marigold, spearmint,ylang-ylang tree, and saponins.

The chemical pesticide can be a single site anti-fungal agent which caninclude but is not limited to benzimidazole, a demethylation inhibitor(DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine,hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outsideinhibitor, quinoline, dicarboximide, carboximide, phenylamide,anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid,hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid(xylylalanine), a demethylation inhibitor selected from the groupconsisting of imidazole, piperazine, pyrimidine and triazole (e.g.,bitertanol, myclobutanil, penconazole, propiconazole, triadimefon,bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole,tebuconazole, tetraconazole), myclobutanil, and a quinone outsideinhibitor (e.g., strobilurin). The strobilurin can include but is notlimited to azoxystrobin, kresoxim-methoyl or trifloxystrobin. In yetanother particular embodiment, the anti-fungal agent is a quinone, e.g.,quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether). Theanti-fungal agent can also be derived from a Reynoutria extract. Thechemical pesticide can also be a multi-site non-inorganic, chemicalfungicide. For example, the chemical fungicide can be chloronitrile,quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate,chloralkythios, phenylpyridin-amine, or cyano-acetamide oxime.

Nematicides can include, but are not limited to, avermectin nematicides(e.g., abamectin); botanical nematicides (e.g., carvacrol); carbamatenematicides (e.g., benomyl carbofuran, carbosulfan, cloethocarb); oximecarbamate nematicides (e.g., alanycarb, aldicarb aldoxycarb, oxamyltirpate); fumigant nematicides (e.g., carbon disulfide, cyanogen,1,2-dichloropropane, 1,3-dichloropropene, dithioether, methyl bromide,methyl iodide, sodium tetrathiocarbonate); organophosphorus nematicides,which includes, but are not limited to, organophosphate nematicides(e.g., diamidafos, fenamiphos, fosthietan, phosphamidon);organothiophosphate nematicides (e.g., cadusafos, chlorpyrifos,dichlofenthion dimethoate ethoprophos, fensulfothion, fosthiazate,heterophos, isamidofos, isazofos, phorate, phosphocarb, terbufos,thionazin, triazophos); phosphonothioate nematicides (e.g., imicyafos,mecarphon); and other nematicides (e.g., acetoprole, benclothiaz,chloropicrin, dazomet, DBCP, DCIP, fluensulfone, furfural, metam, methylisothiocyanate, xylenols, spirotetramat).

The compositions disclosed herein can also be used in combination withother growth promoting agents such as synthetic or organic fertilizers(e.g., di-ammonium phosphate in either granular or liquid form), compostteas, seaweed extracts, plant growth hormones such as IAA (indole aceticacid) used in a rooting hormone treatment for transplants either aloneor in combination with plant growth regulators such as IBA (indolebutyric acid) and NAA (naphthalene acetic acid), and growth promotingmicrobes, such as Bacillus spp., Pseudomonads, Rhizobia, and Trichodermaspp.

Furthermore, the compositions can be used in combination withseed-coating agents. Such seed coating agents can include, but are notlimited to, ethylene glycol, carboxymethyl cellulose, methyl cellulose,polyethylene glycol, chitosan, carboxymethyl chitosan, peat moss, resinsand waxes. The compositions can be applied using methods known in theart. Specifically, these compositions can be applied to and aroundplants or plant parts. Plants are to be understood as meaning in thepresent context all plants and plant populations such as desired andundesired wild plants or crop plants (including naturally occurring cropplants). Crop plants can be plants which can be obtained by conventionalplant breeding and optimization methods or by biotechnological andgenetic engineering methods or by combinations of these methods,including the transgenic plants and including the plant cultivarsprotectable or not protectable by plant breeders' rights. Plants includeall parts and organs of plants above and below the ground, such asshoot, leaf, flower and root, examples which may be mentioned beingleaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds,roots, tubers and rhizomes. The plants include, but are not limited to,harvested material, and vegetative and generative propagation material,for example cuttings, tubers, rhizomes, offshoots and seeds.

The compositions, cultures and supernatants and pesticidal compounds setforth above can be used particularly to modulate infestation of cornrootworm larvae on a plant, plant seed, plant part, plant roots.seedling or substrate (e.g., soil, sand, loam, clay) for growing saidplant, particularly a corn plant, cucurbits (e.g., cucumbers, melons,pumpkins, squash, gourds, etc.), wheat, barley, oats, rye, sorghum,beans and legumes, peas, potato, sweet potato, soy, oilseed rape,tomato, aubergine, lettuce, pepper, sunflower, and ornamental plantssuch as chrysanthemum as well as other plants such as red root pigweed(Amaranthus retroflexus), goosegrass (Eleusine indica) weeping lovegrass(Eragrostis curvula), sand lovegrass (Eragrostis trichodes), rhodesgrass (Chloris gayana), shattercane (Sorghum drummondii), johnsongrass(Sorghum halepense), maize (Zea mays), sandbur (Cenchrus tribuloides),large crabgrass (Digitaria sanquinalis), barnyard grass, (Echinochloacrus-galli), woolly cupgrass (Eriochloa villosa), witchgrass (Panicumcapillare), foxtail millet (P. italicum), Prosso millet (P. miliaceum),switchgrass (P. virgatum), giant foxtail (Setaria faberi), yellowfoxtail (S. pumila), bristly foxtail (S. vericillatta), Green foxtail(S. viridis), texas panicum (Urochloa texana), Redtop (Agrostisgigantean), oat (Avena sativa), reed canarygrass (Phalaris arundinacea),downy brome (Bromus tectorum), Orchardgrass (Dactylis glomerata),western wheatgrass (Pascopyrum smithii), spring wheat “Russ” (Triticumaestivum)

The present disclosure also relates to the use of Burkholderia sp. tomodulate members go the genus Diabrotica (rootworm) including Diabroticabalteata (banded cucumber beetle), Diabrotica barberi (northern cornrootworm), Diabrotica beniensis, Diabrotica cristata, Diabroticacurvipustulata, Diabrotica dissimilis, Diabrotica elegantula, Diabroticaemorsitans, Diabrotica graminea, Diabrotica hispanolae, Diabroticalemniscata, Diabrotica linsleyi, Diabrotica longicornis, Diabroticamilleri, Diabrotica nummularis, Diabrotica occlusa, Diabrotica porracea,Diabrotica scutellata, Diabrotica speciosa (cucurbit beetle orchrysanthemum beetle), Diabrotica tibialis, Diabrotica trifasciata,Diabrotica undecimpunctatahowardi (southern corn rootworm, a.k.a spottedcucumber bettle), Diabrotica undecimpunctata tenella (western cucumberbeetle), Diabrotica undecimpunctata undecimpunctata (western spottedcucumber beetle), Diabrotica virgifera virgifera (western cornrootworm), Diabrotica virgifera zea (Mexican corn rootworm), Diabroticaviridula, Diabrotica significata (three-spotted cucumber beetle)

In a particular embodiment, infestation of corn rootworm larvae ismodulated by modulating mean mortality of said larvae at a particularlocation, particularly at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80% and more particularly at least about 90% within at least followingapplication of said compositions, cultures and supernatants andpesticidal compounds set forth above.

Application of an effective controlling amount of Burkholderia sp. forDiabrotica spp. is also provided herein. A substantially pure culture,whole cell broth, supernatant, filtrate, extract or compound of thebacterial strain can be applied, alone or in combination with anothersubstance, in an effective pest control or pesticidal amount. An“effective amount” is defined as the quantity of microorganism cells,supernatant, whole cell broth, filtrate, cell fraction or extract,metabolite and/or compound alone or in combination with anotherpesticidal substance that is sufficient to modulate plant corn rootworminfestation. The amount that will be within an effective range can bedetermined by laboratory or field tests by those skilled in the art. Inan embodiment of the invention, when the composition is applied directlyto the seed, the effective amount is a concentration of about 0.01%-30%of the active ingredient Burkholderia sp. per 100 g of seed. In anotherembodiment, the effective amount is a concentration of about 0.05%-25%,or about 0.1%-20%, or about 0.5%-15%, or about 1%-10%, or about 2%-5% ofthe active ingredient per 100 g of seed. In yet another embodiment, theeffective amount is a concentration of about 0.5% to 1% of the activeingredient per 100 g of seed. In another embodiment of the invention,when the composition is applied to the soil by, for example, in furrow,the effective amount is about 0.1-50 oz. of the active ingredient per1000 ft row. In other embodiments, the effective amount for soilapplication is about 1-25 oz. of the active ingredient per 1000 ft row.In yet another embodiment, the effective amount is about 2-20 oz, orabout 3-15 oz, or about 4-10 oz, or about 5-8 oz, of the activeingredient per 1000 ft row. Yet in another embodiment, the effectiveamount is about 14 or 28 oz./1000 ft row.

The compositions, substantially pure culture, whole cell broth,supernatant, filtrate extract or compounds set forth above can modulatecorn root worm infestation by a number of mechanisms. These include butare not limited to exterminating the plant parasitic at various stagesof the life cycle (eggs, larvae or adult), affecting the motility of theplant parasitic, and/or reducing the number and/or viability of eggsthat are laid by the parasitic.

The said formulated product can be used alone or sequentially with oneor more other components as set forth above, such as growth promotingagents in a tank mix or in a program (sequential application calledrotation) with predetermined order and application interval during thegrowing season.

Contact of the plants with the compositions set forth above can becarried out directly or by allowing the compositions to act on theirsurroundings, habitat or storage space by, for example, immersion,spraying, evaporation, fogging, scattering, painting on, injecting orsoil amendment. In the case that the composition is applied to a seed,the composition can be applied to the seed as one or more coats prior toplanting the seed using one or more coats using methods known in theart.

The compositions comprising Burkholderia sp. can be applied by root dipat transplanting, specifically by treating a fruit or vegetable with thecomposition by dipping roots of the fruit or vegetable in a suspensionof said composition (about 0.25 to about 1.5% and more particularlyabout 0.5% to about 1.0% volume by volume) prior to transplanting thefruit or vegetable into the soil.

Alternatively, the composition can be applied by drip or otherirrigation system. Specifically, the composition comprising Burkholderiasp. can be injected into a drip irrigation system. In a particularembodiment, the composition may be applied at a rate of about 11 toabout 4 quarts per acre.

In yet another embodiment, the composition can be added as an in-furrowapplication. Specifically, the composition can be added as an in-furrowspray at planting using nozzles calibrated to deliver a total output of2-6 gallons/acre, or at outputs of up to 20 gallons per acre. Nozzlesare placed in the furrow opener on the planter so that the pesticideapplication and seed drop into the furrow are simultaneous. The mixturesset forth above and, where appropriate, a solid or liquid adjuvants areprepared in known manner. For example, the mixtures can be prepared byhomogeneously mixing and/or grinding the active ingredients withextenders such as solvents, solid carriers and, where appropriate,surface-active compounds (surfactants). The compositions can alsocontain further ingredients such as stabilizers, viscosity regulators,binders, adjuvants as well as fertilizers or other active ingredients inorder to obtain special effects.

Yet in another embodiment, the compositions from Burkholderia sp. canincrease plant yield, such as measured in bu/A at 15% moisture for Zeamays, while providing CRW infestation modulation.

In one embodiment, the present microbial components can be formulated asdusts or powders, water dispersible formulations, emulsions, solutions,baits and fumigants or aerosols. In addition to the active ingredient,these formulations include carriers, solvents, emulsifiers, wettingagents, dispersing agents, stickers, UV screens, and other inertingredient to maintain stability and shelf-life of the activeingredient. These additional ingredient preferably are selected fromthose listed in EPA Inert List 4a(www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventionalformulations and 4b (www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf)for organic formulations.

EXAMPLES

The composition and methods set forth above will be further illustratedin the following, non-limiting Examples. The examples are illustrativeof various embodiments only and do not limit the claimed inventionregarding the materials, conditions, weight ratios, process parametersand the like recited herein.

Example 1

Four field trials were established in the Midwestern United States totest the efficacy of several insecticide treatments in protecting cornroots from damage by corn rootworm larvae. Experimental trial design ineach case was a randomized complete block with 6 replications pertreatment. Individual plots were 5-10′ wide×25-30′ in length. Cornhybrids were selected for the trials and could be either conventional(non-GMO traited) hybrids or GMO-traited hybrids containing, at aminimum, one or more corn rootworm control traits (Table 1). The betweenrow spacing in each case was 30″ thus individual plots were made up ofeither 2 or 4 rows per trial. Planting depth was 1-2″ and plantingpopulation per acre was between 28,700 and 34,000 seeds per acre. Normalagronomic practices regarding tillage, fertility, non-target pestcontrol and crop production were followed in each trial.

TABLE 1 Ref. # Corn Hybrid Corn Traits in Seed Seed Trt. in Bag Trial 1Blue River 56M30 None None Trial 2 DKC52-43VT3 AGP (2), CRW (1), RRComprehensive Trial 3 Pioneer 1023AM AGP (2), RR, LL Comprehensive Trial4 DKC Smart Stax AGP (3), CRW (2), RR, Comprehensive LL AGP = traits forabove-ground pests like European Corn Borer, Corn earworm, etc. Numberin parentheses indicates number of traits. CRW = Corn Rootworm RR =Roundup Ready (glyphosate) herbicide tolerant LL = Liberty Link(gluphosinate) herbicide tolerance ‘Comprehensive’ seed treatmentgenerally includes one or more fungicides, insecticides and possiblyeven nematicide components to protect seed from feeding from diseasesand below-ground pests like wireworm, seed corn maggot, etc

Treatments included: an untreated check; MBI-206 (also known asBurkholderia A396); MBI-206BM3SE1 (a.k.a Burkholderia A396 or Venerate®XC, also see FIGS. 1-3) applied at 14 or 28 ounces of formulated productper 1000 row ft. in-furrow; and an in-furrow competitive (commercial)standard (Capture LFR @ 0.4-0.49 oz/1000 row feet or Lorsban 4E @ 8oz/1000 row feet). A subsample of the selected hybrid seed were sent toMarrone Bio Innovations in Davis, Calif. for seed coating in thelaboratory. These treated seed were then returned for planting.

Root feeding damage by corn rootworm larvae was assessed by digging rootsystems 53-86 days after planting, washing them, and measuring themusing the Iowa State University 0-3 Corn Rootworm Damage scale where 0,1, 2 & 3 equate to the number of root nodes completely chewed off bycorn rootworm larvae; i.e. the greater the number the more intense thefeeding damage. Yield measurements (bushels/acre at 15% moisture) wheretaken occurred at harvest.

Results

In ¾ studies there was heavy CRW pressure and feeding damage. In 1 ofthose locations seed was planted having CRW traits and still sustainedheavy CRW damage. This indicates resistance to the CRW traits in thishybrid. Overall the product treatments performed better than the UTC(un-treated control). There did not appear to be a consistent rateeffect for the ones applied in-furrow. Table 2 summarized the results,where SDTR=seed treatment. Trial 2 has light CRW (corn Rootworm)infestation. The Table indicates Burkholderia A396's activity againstCRW, especially under heavy CRW pressure.

TABLE 2 Avg. 3 trials with Trial Trial Trial Trial Avg. HeavyTreatment/Rate Type 1 2 3 4 All CRW Untreated Check — 2.24 0.32 2.252.12 1.73 2.20 MBI-206 BM3SE1 In- 0.81 0.27 0.56 0.72 0.59 0.70 @ 14oz/1000 row Furrow ft. MBI-206 BM3SE1 In- 1.15 0.21 1.05 0.67 0.77 0.96@ 28 oz/1000 row Furrow ft. MBI-206 Low Rate SDTR 1.06 0.22 0.82 0.720.71 0.87 MBI-206 High Rate SDTR 1.18 0.25 0.69 0.56 0.67 0.81Commercial In- 0.19 0.23 0.88 0.44 0.44 0.50 Standard Furrow

FIGS. 1-3 (trials 2-4) demonstrates that while Burkholderia A396 (206)modulates CRW infestation, it can also increase overall Zea mays yieldin Bu/A. Almost all of the IF applications of Burkholderia A396 presentsstatistically the same, or statistically more, than the chemical oruntreated control.

Example 2 Formulation and Application of Pesticidal Toxins (206) asPowders and Dusts

These are simple formulations that usually contain 0.1-25% of the activeingredient Burkholderia A396 (206). However, more active ingredient canbe used depending on the potency and particular application. Thepesticide toxin is mixed with a solid carrier, which can be of smallparticle size. Solid carriers can include: silicate clays (e.g.,attapulgite, bentonites, volcanic ash, montmorillionite, kaolin, talc,diatomites, etc.), carbonates (e.g., calcite, dolomite, etc), synthetics(precipitated silica, fumed silica, etc.), ground botanicals (e.g., corncob grits, rice hulls, coconut shells, etc.), organic flour (e.g.,Walnut shell flour, wood bark, etc.) or pulverized mineral (e.g.,Sulphur, diatomite, tripolite, lime, gypsum talc, pyrophyllite, etc.).The inert ingredients used in dust formulations can also come from thoselisted in EPA Inert List 4a(www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventionalformulations and 4b (www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf)for organic formulations. Small particle size can be achieved by mixingthe active with the carrier and pulverizing in a mill. Dusts are definedas having a particle size less than 100 microns and with increase inparticle size the toxicity of the formulation decreases. In theselection of a dust formulation its compatibility, fineness, bulkdensity, flow ability, abrasiveness, absorbability, specific gravity andcost should be taken into consideration.

Formulation Formulation Formulation Formulation Formulation components AB C D Active ingredient 0.65  5 10 25 Talc 90 Kaolin or other 95 clay

A dust formulation can also be prepared from a dust concentrate (e.g.,40% active ingredient, 5% stabilizer, 20% silica, 35% magnesiumcarbonate) added at 1-10% to a 1:1 organic filler/talc combination. Thedust formulation is used as a contact powder (CP) or tracking powder(TP) against crawling insects. A dust formulation with high flowabilitycan be applied by pneumatic equipment in greenhouses.

Example 3 Formulation of Pesticidal Toxins as Granules or Pellets

The present microbial component (206, a.k.a. Burkholderia A396) isapplied in liquid form to coarse particles of porous material (e.g.,clay, walnut shells, vermiculite, diatomaceous earth, corn cobs,attapulgite, montmorillioinite, kaolin, talc, diatomites, calcite,dolomite, silicas, rice hulls, coconut shells, etc.). The granules orpellets can be water dispersible, and can be formed by extrusion (forpesticidal actives with low water solubility), agglomeration or spraydrying. Granule can also be coated or impregnated with a solvent basedsolution of the pesticidal toxin. The carrier particles can be selectedfrom those listed in EPA Inert List 4a(www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventionalformulations and 4b (www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf)for organic formulations. The active can be absorbed by the carriermaterial or coat the surface of the granule. Particle size can vary from250 to 1250 microns (0.25 mm to 2.38 mm) in diameter. The formulationsusually contain 2 to 10 percent concentration of the toxicant. Thegranules are applied in water or whorls of plant or to soil at the rateof 10 kg/ha. Granular formulations of systemic insecticides are used forthe control of sucking and soil pest by application to soil. Whorlapplication is done for the control of borer pests of crops such assorghum, maize and sugarcane, etc. These types of formulations reducedrift and allow for slower release of the pesticide active.

Granular pesticides are most often used to apply chemicals to the soilto control weeds, fire ants, nematodes, and insects living in the soilor for absorption into plants through the roots Granular formulationsare sometimes applied by airplane or helicopter to minimize drift or topenetrate dense vegetation. Once applied, granules release the activeingredient slowly. Some granules require soil moisture to release theactive ingredient. Granular formulations also are used to control larvalmosquitoes and other aquatic pests. Granules are used in agricultural,structural, ornamental, turf, aquatic, right-of-way, and public health(biting insect) pest control operations.

Application of granular formulations is common in pre-emergenceherbicides or as soil insecticides for direct application andincorporation into soli or other solid substrates where plants grow.Granules or pellets can also be applied in-furrow. Granules are commonlyused for application top water, such as in flooded rice paddies.

A typical granule formulation includes (% w/w) 1-40% active ingredient,1-2% stabilizer, 0-10% resin or polymer, 0-5% surfactant, 0-5% binderand is made up to 100% with the carrier material.

Example 4 Formulation of Pesticidal Toxins as Wettable Powders

Wettable powder is a powdered formulation which yields a rather stablesuspension when diluted with water. It is formulated by blending thetoxicant with diluents such as attapulgite, a surface active agent andan auxiliary material such as sodium salts of sulfo acids. Sometimesstickers are added to improve retention on plants and other surfaces.Wettable powders can be prepared by mixing the pesticidal toxin (10-95%)with a solid carrier, plus 1-2% of a surface-active agent to improvesuspensibility. The overall composition of the formulation will includethe active ingredient in solid form (5.0-75%), an anionic dispersant andan anionic/nonionic wetting agent.

A typical example of wettable powder formulation will include 10-80%active ingredient, 1-2% wetting agents (e.g., benzene sulphonates,naphthalene sulphonates, aliphatic suplhosuccinates, aliphatic alcoholetoxylates, etc.), 2-5% dispersing agent (e.g., lignosulphonates,naphthalene sulphonate-formaldehyde condensates, etc.), 0.1-1%antifoaming agent, made up to 100% with an inert filler or carrier(e.g., diatomaceous earth, silica, etc.).

Example 5 Formulation of Pesticidal Toxins as Emulsifiable Concentrates

It is a concentrated pesticide formulation containing organic solventand a surfa-active agent to facilitate emulsification with water. WhenEC formulations are sprayed on the plant parts, the solvent evaporatesquickly leaving a deposit of toxicant from which water also evaporates.Some of the emulsifying agents in insecticide formulations are alkalinesoaps, organic amines, sulfates of long chain alcohols and materialssuch as alginates, carbohydrates, gums, lipids and proteins. Emulsifyingagents can be selected from those listed in EPA Inert List 4a(www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventionalformulations and 4b (www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf)for organic formulations.

Example 6 Formulation of Pesticidal Toxins as Solutions

It is a concentrated liquid pesticide formulation (206) that can be useddirectly or require diluting (soluble concentrates). Solubleconcentrates and solutions are water or solvent based mixture withcomplete miscibility in water.

A typical example of a solution concentrate formulation include 20-70%active ingredient, 5-15% wetting agent, 5-10% antifreeze, and is made upto 100% with water or a water miscible solvent.

Depending on the nature and stability of the pesticidal toxin, asolution formulation might also include thickeners, preservatives,antifoam, pH buffers, UV screens, etc.

Example 7 Formulation of Pesticidal Toxins in Fertilizers Mixtures.

The mixture generally constitutes addition of a granular insecticide tochemical fertilizer or spreading of insecticide directly on to thefertilizer. They are applied at the regular fertilizing time and provideboth plant nutrients and control of soil insects. Urea 2% solution ismixed with compatible insecticidal emulsions and sprayed for supply ofnitrogen to the plant and for realizing effective pest control. Manypesticides are rapidly broken down when mixed with fertilizers.

Example 8 Formulation of Pesticidal Toxins as Poison Baits.

The poison baits consist of a base or carrier material attractive to thepest species and a chemical toxicant in relatively small quantities. Thepoison baits are used for the control of fruit flies, chewing insects,wireworms, white grubs in the soil, household pests rats in the fieldand slugs. This method is ideal under conditions where spray applicationis rather difficult. For successful poison baiting the attractiveness,palatability, toxicity, stability and physical condition of the baits,as also the time, place and method of exposure must be considered. Thecommon base used in dry baits is wheat bran moistened with water andmolasses an attractant and a toxicant. For the control of fruit suckingmoths fermenting sugar solution or molasses with a toxicant is used.

Example 9

Formulation of Pesticidal Toxins into Seed Treatments

Seed treatments include the application of the pesticidal toxins, orother bioactive, antagonistic or symbiotic agents to the surface of thesee prior to sowing. The pesticidal toxins, proteins, compound of theinvention can be applied to seeds as dry powders, slurried powders orsprayed on the seed before planting.

The pesticidal toxins can be formulated for seed treatments in any ofthe following modes: dry powder, water slurriable powder, liquidsolution, flowable concentrate or emulsion, emulsion, microcapsules,gel, water dispersible granules.

In the case of a dry powder, the active ingredient in formulatedsimilarly to a wettable powder, but with the addition of a stickingagent, such as mineral oil, instead of a wetting agent. For example: OneKg of purified talc powder (sterilized for 12 h), 15 g calciumcarbonate, and 10 g carboxy methyl cellulose were mixed under asepticconditions following the method described by Nandakumar et al (2001).Protein, nucleic acid suspensions or organisms expressing these weremixed in a 1:2.5 ratio (suspension to dry mix) and the product was shadedried to reduce moisture content to 20-35%.

There are several ways in which seed can be treated with theformulations:

Priming: seeds are soaked in double the volume of sterile distilledwater containing bacterial/protein/nucleic acid suspensions or talcformulation (dry formulation) (4-10 g kg-1 of seed, depending on seedsize) and incubated at 25±2° C. for 12-24 h. The suspension is drainedoff and the seeds are dried under shade for 30 min and used for sowing.

Coating: for seed coating different stickers are used, within them,methyl cellulose, alginate, carrageenan and polyvinyl alcohol. These aremixed at percentages between 1-10% as water based solutions and storedat room temperature before application to the seeds. Seeds are soaked insticker solution (3 ml/100 seeds) for 15 min, scooped out and mixed withorganic matter (1.5 g/100 seeds) in plastic bags and shaken vigorously.This process can be automated using a seed coating machine.

What is claimed is:
 1. A method for modulating infestation of Diabrotica(corn rootworm) in a location where modulation is desired comprisingapplying an effective amount of (a) a culture, suspension or whole cellbroth comprising a strain of Burkholderia sp., or supernatant, filtrate,cell fraction, and/or extract derived from said culture, suspension orwhole cell broth, and (b) optionally at least one of a carrier, diluentor adjuvant, to modulate said infestation of corn rootworm at saidlocation.
 2. The method according to claim 1, wherein the location wheremodulation is desired is on a plant, plant seed, plant roots, plantpart, seedling or substrate for growing said plant.
 3. The methodaccording to claim 1, wherein said Burkholderia sp. is Burkholderia A396strain.
 4. The method according to claim 1, wherein said Burkholderiasp. has the identifying characteristics of (NRRL Accession No. B-50319).5. The method according to claim 1 wherein said Diabrotica (cornrootworm) is Western or Northern Corn Rootworm.
 6. The method accordingto claim 1, wherein said Diabrotica (corn rootworm) is modulated bymodulating the mortality of said corn rootworm.
 7. The method accordingto claim 1, wherein the mortality of Diabrotica (corn rootworm) ismodulated and wherein there is a mortality of corn rootworm of at leastabout 50% at said location.
 8. The method according to claim 1, whereinthe wherein the mortality of corn rootworm is modulated and whereinthere is a mortality of Diabrotica (corn rootworm) of at least about 50%following application of said culture, suspension or whole cell brothcomprising a strain of Burkholderia sp., or supernatant, filtrate, cellfraction, extract and/or one or more compounds derived from saidculture, suspension or whole cell broth and (b) at least one of acarrier, diluent or adjuvant at said location.
 9. The method accordingto claim 1, which further comprises applying another natural orartificial insecticidal substance.
 10. The method according to claim 1,wherein the infestation of Diabrotica (corn rootworm) is modulated bymodulating the rate of hatching of eggs.
 11. The method according toclaim 1, wherein said culture, suspension or whole cell broth comprisinga strain of Burkholderia sp., or supernatant, filtrate, cell fraction,and/or extract derived from said culture, suspension or whole cell brothfurther comprise at least one of a carrier, diluent or adjuvant isformulated into a composition.
 12. A method for modulating plant yieldand Diabrotica infestation comprising applying an effective amount of aculture, suspension or whole cell broth comprising a strain ofBurkholderia sp., or supernatant, filtrate, cell fraction, and/orextract derived from said culture, suspension or whole cell broth to aplant or plant part.